Inhibition of the mutagenicity of aromatic amines by the plant flavonoid (+)-catechin

Addition of the plant phenolic flavonoid (+)-catechin to rat liver microsomes inhibited the mutagenicity of the aromatic amines 2-aminofluorene and 4-aminobiphenyl in the Ames test. Similarly, (+)-catechin decreased the mutagenicity of N-hydroxy-4-amino-biphenyl, the proximate carcinogen, but, in contrast, had no effect on the mutagenicity of other direct-acting carcinogens such as N-methyl-N'-nitro-N-nitrosoguanidine and 9-aminoacridine. In vitro addition of (+)-catechin gave rise to a dose-dependent inhibition of the cytochrome P-450-dependent benzphetamine N-demethylase and ethoxyresorufin O-deethylase activities. This was achieved by impairment of the electron flow from the reduced pyridine nucleotide to the cytochrome. However, administration of (+)-catechin to rats had no effect on the in vitro mixed-function oxidase activities. It is concluded that the (+)-catechin inhibits the mutagenicity of aromatic amines in the Ames test by interfering with their cytochrome P-450-dependent bioactivation and by direct interaction with the proximate carcinogen, but the former mechanism is unlikely to occur in vivo because the high doses of the flavonoid required are not achieved.     

A rationale for the non-mutagenicity of 2- and 3-aminobiphenyls

Of the three isomeric forms of aminobiphenyl, only 4-aminobiphenyl is an established carcinogen while the 2-isomer is considered as a non-carcinogen and 3-aminobiphenyl is at best described as a weak carcinogen. In the present studies we investigated the mutagenicity of these three compounds, their N-hydroxy derivatives and their nitrosoderivatives in the Ames test using the Salmonella typhimurium strains TA98 and TA100. The studies were performed both in the absence and presence of an activation system derived from the liver of rats pretreated with Aroclor 1254. Of the three isomers only 4-aminobiphenyl exhibited mutagenicity and only in the presence of an activation system. N-Hydroxy-4-aminobiphenyl was a potent direct mutagen in both bacterial strains, N-hydroxy-2-aminobiphenyl was mutagenic in only TA100 while N-hydroxy-3-aminobiphenyl displayed mutagenicity in neither strain. Both 2- and 3-nitrosobiphenyls were direct mutagens in strain TA100. These findings suggest that the weak carcinogenicity of 3-aminobiphenyl may be attributed to the lack of genotoxicity of its N-hydroxyderivative, whereas in the case of 2-aminobiphenyl it may be due to the inability of the hepatic preparations to catalyse its N-hydroxylation, which is in agreement with published in vivo metabolic studies. It is interesting that of the three isomers only 2-aminobiphenyl is non-planar, forming a dihedral angle of 40 degrees, and this may preclude it from acting as a substrate of the P-450I family of haemoproteins, which selectively catalyses the N-hydroxylation of many aromatic amines including 4-aminobiphenyl.     

Metabolic activation of phenacetin and phenetidine by several forms of cytochrome P-450 purified from liver microsomes of rats and hamsters

Metabolic activation of phenacetin by liver microsomes proceeds via both phenetidine and N-hydroxyphenacetin to direct-acting mutagens, i.e., N-hydroxyphenetidine and p-nitrosophenetole. Five different molecular species of cytochrome P-450 have been purified from liver microsomes of drug-pretreated Wistar rats or Syrian hamsters and their abilities to activate phenetidine and phenacetin were compared using reconstituted microsome systems. High-spin forms of cytochrome P-450 purified from 3-methylcholanthrene-pretreated rats (MC-P-448-H) or hamsters (P-488 ham-II) showed higher catalytic activity for N-hydroxylation of phenetidine than three other low-spin forms of cytochrome P-450 purified from the same animals or from phenobarbital-pretreated rats. MC-P-448-H and P-488 ham-II required the presence of cytochrome b5 for their maximum activities in the reconstituted system. The five forms of cytochrome P-450, however, exhibited no measurable activity for N-hydroxylation of phenacetin either with or without cytochrome b5. The mutagenicity of phenacetin and phenetidine toward Salmonella typhimurium TA100 was generated when the reconstituted microsomes containing MC-P-488-H or P-488 ham-II were used as activating enzymes. From these results, it was suggested that high-spin forms of cytochrome P-450 (MC-P-448-H and P-448 ham-II) played an important role in the metabolic activation of phenacetin to the direct-acting mutagens.     

Metabolic activation of a protein pyrolysate promutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline by rat liver microsomes and purified cytochrome P-450

The enzymatic activation of a promutagenic pyrolysate, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MeIQx), was studied using the Ames mutagenesis test system.The enzyme catalyzing the mutagenic activation of MeIQx is mainlylocalized in the microsomal fraction. A large number of revertantswas observed in the presence of hepatic microsomes obtainedfrom 3-methylcholanthrene (3-MC)- or polychlorinated biphenyl(PCB)-treated rats but only a minimal number with the hepaticmicrosomes from untreated or phenobarbital (PB)-treated rats.In addition, the microsomal activation was reduced efficientlyby known inhibitors of cytochrome P-450- mediated reactionssuch as 7,8-benzoflavone, ellipticine and flavone. Among fiveforms of purified rat cytochrome P-450, the highest sp. act.(no. of revertants induced/nmol cytochrome P-450) for the activationof MeIQx was observed with a high-spin form of cytochrome P-450,P-448-H, followed by the low-spin form, P-448-L, and to a lesserextent by PB-inducible forms, P-450b and P-450e. P-450-male,which is a main constitutive form of cytochrome P-450 In malerat livers, showed considerable catalysis for the mutagenicactivation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) andMeIQx. These results Indicate that the metabolic activationof MeIQx is catalyzed mainly by two forms of cytochrome P-450,P-448-H and P-488-L, in the livers of PCB- or 3-MC-treated rats,but also that P-450-male may play an important role in the activationin livers of Intact male rats.     

Activation of 3,3'-dichlorobenzidine in rat liver microsomes to mutagens: involvement of cytochrome P-450d

Studies were carried out, using antibodies to specific cytochromeP-450 isozymes, to identify the isozymes involved in the NADPH-dependentactivation of 3,3'-dichlorobenzidine (DCB) by rat hepatic microsomesto mutagens in the Ames test. DCB activation was not affectedby a monoclonal antibody specific for P-450c or by a monoclonalantibody specific for P-450b, but was inhibited 69% by a polyclonalantibody made against P-450d. DCB activation was also inhibited46% by antibody specific for NADPH-cytochrome P-450 reductase.Further, addition of methimazole, a high affinity substratefor the flavin-containing monooxygenase, reduced the residualmutagenicity in the systems containing antibody to P-450d andcytochrome P-450 reductase to 9% and 19%, respectively, of theappropriate control values. It is concluded that P-450d contributesto a majority of the P-450-dependent activation of DCB in hepaticmicrosomes. The results also suggest that the flavin-containingmonooxygenase may contribute to the microsomal activation ofDCB.     

Specificity of rabbit cytochrome P-450 isozymes involved in the metabolic activation of the food derived mutagen 2-amino-3-methylimidazo[4,5-f] quinoline

The involvement of rabbit liver cytochromes P-450 in the activation of the food derived heterocyclic amine mutagen, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), was assessed using the Ames/Salmonella test. The number of revertants induced by IQ per microgram of control rabbit liver microsomes was 1872 +/- 50 (SD, n = 3), and this increased to 3690 +/- 239 when microsomes from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) animals were used as the metabolic activation source. Microsomes from phenobarbital, rifampicin, and acetone pretreated rabbits were less efficient the controls at activating IQ to a mutagen. Cytochrome P-450 Forms 4 and 6, which are induced by TCDD, were found to be efficient activators of IQ to a mutagen in reconstitution experiments. Form 4 was 7.7-fold more active than Form 6 and produced 1702 revertants/0.125 pmol with a 20-min preincubation step in the Ames test. Anti-Form 4 IgG inhibited the activation of IQ in control and TCDD induced microsomes by 78 and 79%, respectively. The contents of cytochrome P-450 Form 4, determined by Western blot analysis, in control and phenobarbital, acetone, rifampicin, and TCDD pretreated microsomes were 0.55 +/- 0.19, 0.63 +/- 0.34, 0.5 +/- 0.27, 0.28 +/- 0.16, and 2.19 +/- 0.43 (n = 3) nmol/mg protein, respectively. A highly significant statistical correlation existed between the capacity of the above microsomes to activate IQ to a mutagen and their cytochrome P-450 Form 4 content (r = 0.96; r2 = 0.92). The content of cytochrome P-450 Form 6 in the above microsomes was also highly correlated with their capacity to activate IQ (r = 0.92; r2 = 0.85). Based on these results and the tissue distribution of cytochrome P-450 Forms 4 and 6, the former obviously contributes most toward the activation of IQ in the liver, whereas Form 6 would be expected to be primarily involved in this process in extrahepatic tissues.     

The mutagenicity of 9-hydroxyellipticine and its induction of cytochrome P-448 activity in rat liver microsomes

Administration of a single dose of the inhibitor of the hepaticmixed function oxidases, 9-hydroxyellipticine (9-OHE), resultedin a marked increase in the cytochrome P-448 catalysed activitiesof ethoxyresorufin O-deethylase, biphenyl 2-hydroxylase andactivation of benzo[a]pyrene to mutagens. In contrast therewas no effect on the cytochrome P-450 catalysed benzphetamineN-demethylase activity. The inductive effect was prevented bysimultaneous administration of the protein synthesis inhibitorscytoheximide and actinomycin D. It is concluded that 9-OHE mayalso act as an inducer of the hepatic microsomal mixed functionoxidases, selectively inducing the synthesis of cytochrome P-448.Therefore, 9-OHE, like other inhibitors of this enzyme system,exhibits a biphasic effect, its inhibitory phase being followedby one of enzyme induction. 9-OHE was a direct mutagen in theSalmonella typhimurium strains TA 98 and TA 100.     

Selective mutagenic activation by cytochrome P3-450 of carcinogenic arylamines found in foods

Heterocyclic arylamines found in cooked foods including fish and beef are potent mutagens and carcinogens. The purpose of this investigation was to determine the specificity of cytochromes P1-450 and P3-450 toward the metabolic activation of these arylamines. We used a novel mutagenicity test system which combined human cells expressing either recombinant cytochrome P1-450 or P3-450 with Salmonella typhimurium to score mutants. Cytochrome P3-450, a single isoform of the cytochrome P-450 supergene family, bioactivated these food mutagens. Cytochrome P1-450 showed little or no activation of these arylamines but was the isoform predominantly responsible for the activation of the aromatic hydrocarbon benzo[a]pyrene-7,8-diol. This assay system should serve to define the specificities of individual cytochromes P-450 in the metabolic activation of carcinogens.     

Metabolic oxidation of the carcinogens 4-aminobiphenyl and 4,4'-methylene-bis(2-chloroaniline) by human hepatic microsomes and by purified rat hepatic cytochrome P-450 monooxygenases

Metabolic N-oxidation and ring-oxidation of carcinogenic arylamines by hepatic cytochromes P-450 are generally regarded as critical activation and detoxification pathways, respectively. Two arylamines with known human exposure, 4-aminobiphenyl (ABP) and 4,4'-methylene-bis(2-chloroaniline) (MOCA), have been examined as substrates for 10 different purified rat hepatic cytochromes P-450 and for human liver microsomal preparations from 22 individuals. Metabolites were analyzed by high-performance liquid chromatography and flow scintillation techniques. As reported for certain other carcinogenic arylamines, the isosafrole-inducible isozyme, P-450ISF-G, had the highest catalytic activity for ABP N-oxidation (13.6 nmol/min/nmol P-450), but P-450BNF-B, P-450UT-A, P-450UT-F, and P-450PB-B also showed appreciable activity. Ring-oxidation of ABP occurred only to a minor extent. In contrast, N-oxidation of MOCA was preferentially catalyzed by the phenobarbital-inducible enzymes, P-450PB-B and P-450PB-D (9.0 and 6.6 nmol/min/nmol P-450, respectively). MOCA ring-oxidation and methylene carbon oxidation showed varied cytochrome P-450 selectivity and accounted for 14 to 79% of total oxidation products. There was a 44-fold variation in rates of ABP N-oxidation in the 22 human liver microsomal preparations, while rates of N-oxidation of MOCA varied only 8-fold. Ring/methylene carbon-oxidation of MOCA accounted for 6-19% of total oxidation products in the case of the human microsomal preparations, whereas ring-oxidation of ABP accounted for less than 7% of total oxidation. In addition, there was a strong correlation (R = 0.90) between rates of ABP N-oxidation and phenacetin O-deethylation, which is considered a human genetic polymorphism. Moreover, both the ABP N-oxidation and phenacetin O-deethylation activities of human liver microsomes showed a good correlation (R = 0.72) with the levels of cytochrome P-450 immunochemically related to rat P-450ISF-G. These data indicate that specific inducible and constitutive cytochromes P-450 are involved in the metabolic activation and detoxification of the carcinogens ABP and MOCA. Therefore, individual profiles of cytochromes P-450, affected by both environmental and genetic factors, may be significant determinants of individual susceptibility to arylamine carcinogenesis.     

Purification and characterization of cytochrome P-450 induced by benz(a)anthracene in mouse skin microsomes

Topical application of benz(a)anthracene to mouse skin elicited a 2-fold increase in cytochrome P-450 content, with accompanying increases in monooxygenase activities such as benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and acetanilide 4-hydroxylation, in the microsomes. A major form of cytochrome P-450 was purified from skin microsomes of mice treated with polycyclic aromatic hydrocarbon. A specific content of 1.95 nmol/mg of protein, which corresponded to 48-fold purification from the microsomes, was observed. The purified protein produced a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a molecular weight of 55,000. Using Western blotting, the band immunochemically cross-reacted with antibody which had been raised against rat liver cytochrome P-450MC-1. The purified preparation efficiently catalyzed benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation when reconstituted with NADPH-cytochrome P-450 reductase. These activities were inhibited by 7,8-benzoflavone as well as anti-cytochrome P-450MC-1 antibody, but not by P-450PB-1 antibody. The results indicate that, in mouse skin microsomes, a cytochrome P-450 induced by benz(a)anthracene is enzymatically and immunochemically similar to rat liver cytochrome P-450MC-1. It is suggested that this enzyme plays an important role in the activation of carcinogenic polycyclic aromatic hydrocarbons.     

Changes in the quantity and activity of cytochrome P-450 isozymes in primary cultured rat hepatocytes

Hepatocytes from male Sprague-Dawley rats pretreated with a cytochrome P-450 inducer, 3-methoxy-4-aminoazobenzene (3-MeO-AAB), 3-methylcholanthrene (MC) or phenobarbital (PB), were cultured in vitro, and changes in the quantity and activity of microsomal cytochrome P-450 isozymes in the cells were determined by means of immunochemical methods and a bacterial mutation test, respectively. The results of enzyme-linked immunosorbent assay using monoclonal antibodies against rat P-450 isozymes revealed that the amount of cytochrome P-450d induced by 3-MeO-AAB or MC declined rapidly during culture and fell to 10 to 15% of the initial value after 24 h. A similar tendency was observed with PB-induced cytochrome P-450b/e. By contrast, cytochrome P-450c in MC-induced hepatocytes declined more slowly than cytochrome P-450d and remained at 45 to 60% of the initial value after 24 h. Similar quantitative changes of the individual cytochrome P-450 isozymes in culture were also observed by immunoblotting using the anti-cytochrome P-450 monoclonal antibodies. Changes in the activities of individual cytochrome P-450 isozymes in hepatocytes by culture were in accordance with the quantitative changes of the cytochromes, as determined by a mutation test using Salmonella typhimurium TA 98 and carcinogenic aromatic amines. These results indicate that microsomal cytochrome P-450c in primary cultured rat hepatocytes is more stable in culture, in terms of both quantity and activity, than cytochrome P-450d and P-450b/e.     

Covalent binding to DNA and mutagenicity of 2,4-diaminotoluene metabolites produced by isolated hepatocytes and 9000 g supernatant from Fischer 344 rats

Primary cultures of hepatocytes from adult male F344 rats wereused to investigate the activation of the hepatocarcinogen 2,4-diaminotoluene(2,4-DAT) to metabolites which bound covalently to DNA. Covalentbinding of 2,4-DAT to DNA was significantly greater than thatof a non-carcinogenic isomer, 2,6-DAT. Treatment of male ratswith 5,6-benzo-flavone (BNF) an inducer of cytochrome P-450cand P-450d, had no effect on the binding of 2,4-DAT to DNA ofhepatocytes from these animals. However, treatment of hepatocytesin vitro with metyrapone or piperonyl butoxide, two generalinhibitors of P-450 enzymes, inhibited the binding of 2,4-DATto DNA by {small tilde}80–85%. Two inhibitors of sulfation,pentachlorophenol and 2,5-dichloro-4-nitrophenol, also inhibitedDNA binding in hepatocytes from both BNF-induced (91 and 85%respectively) and control rats (82 and 41% respectively), indicatingthat suffation may also be required. 2,4-DAT was a more potentmutagen than 2,5- or 2,6-DAT in the Ames Salmonella mutagenesisassay using hepatic S9 fractions from F344 rats as an activatingsystem. In contrast to DNA binding, activation of 2,4-DAT tomutagens by S9 fractions from BNF treated rats was greater thanthat by S9 from control rats. The present study shows that 2,4-DATis activated by hepato cytes of F344 rats to products whichbind covalently to DNA. Both cytochrome P-450 and sulfationappear to be involved in the activation.     

Biotransformation of N,N',N'-triethylenethiophosphoramide: oxidative desulfuration to yield N,N',N'-triethylenephosphoramide associated with suicide inactivation of a phenobarbital-inducible hepatic P-450 monooxygenase

Oxidative metabolism of the polyfunctional alkylating agent N,N',N'-triethylenethiophosphoramide (thio-TEPA) was studied in isolated rat liver microsomes and purified, reconstituted cytochrome P-450 (P-450) enzyme systems in order to elucidate the pathways of drug oxidation and to identify the possible contributions of individual P-450 enzymes to the bioactivation of this chemotherapeutic agent. Rat liver microsomes were found to catalyze conversion of thio-TEPA to its oxo metabolite, N,N',N'-triethylenephosphoramide (TEPA), in a P-450-dependent reaction that was markedly stimulated by prior in vivo treatment with drug inducers of hepatic P-450 subfamily IIB (phenobarbital), but not by pretreatment with inducers of P-450 subfamilies IA (beta-naphthoflavone) or IIE (isoniazid). Thio-TEPA depletion and TEPA formation catalyzed by phenobarbital-induced liver microsomes were both inhibited by greater than 90% by antibodies selectively reactive with P-450 PB-4 (gene product IIB1), the major phenobarbital-inducible rat liver microsomal P-450 form, but not by antibodies inhibitory toward 7 other rat hepatic P-450s. Oxidation of thio-TEPA to TEPA was also catalyzed by purified P-450 PB-4 (Km (app) 19 microM; Vmax (app) = 11 mol thio-TEPA metabolized/min/mol P-450 PB-4) following reconstitution of the cytochrome with NADPH P-450 reductase in a lipid environment. Metabolism of thio-TEPA by P-450 PB-4 was associated with a suicide inactivation of the cytochrome characterized by kinactivation = 0.096 min-1, KI = 24 microM, and a partition ratio of 136 +/- 28 (SD) mol thio-TEPA metabolized/mol P-450 inactivated. The thio-TEPA metabolite TEPA, however, did not inactivate the cytochrome, nor was it subject to further detectable metabolism. In microsomal incubations, metabolism of thio-TEPA led to the inactivation of P-450 PB-4 (steroid 16 beta-hydroxylase) as well as P-450 IIIA-related enzymes (steroid 6 beta-hydroxylase) and the P-450-independent enzyme steroid 17 beta-hydroxysteroid:NADP+ 17-oxidoreductase, as demonstrated by use of the P-450 form-selective steroidal substrate androst-4-ene-3,17-dione. In contrast, little or no inactivation of microsomal P-450 IIA-related enzymes (steroid 7 alpha-hydroxylase) or microsomal NADPH P-450 reductase was observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Species Difference among Experimental Rodents in the Activity and Induction of Cytochrome P-450 Isozymes for Mutagenic Activation of Carcinogenic Aromatic Amines

The expressions of hepatic microsomal cytochrome P-450 isozymes in male rats, mice, hamsters and guinea pigs were studied comparatively with or without an ip injection of a cytochrome P-450 inducer. The activity and quantity of microsomal cytochrome P-450 isozymes were determined respectively by a bacterial mutation assay with Salmonella typhimurium TA98 and immunochemical assays using monoclonal antibodies against rat cytochrome P-450 isozymes. 3-Methoxy-4-aminoazobenzene (3-MeO-AAB), 2-amino-3-methyl-9 H -pyrido[2,3- b ]indole acetate (MeAαC) and 3-methylcholanthrene were used as cytochrome P-450 inducers, and 7 carcinogenic aromatic amines including 3-MeO-AAB and MeAαC were used as substrates for the mutation assay. By means of these assays, we examined the species differences among rodents in the activity and induction rate of hepatic cytochrome P-450 isozymes responsible for the mutagenic activation of carcinogenic aromatic amines.     

Biochemical effects of some derivatives of dipyrido[1,2-a:3,''2''-d]imidazole related to protein pyrolysates on rat liver microsomes

Several derivatives of dipyrido [1,2-a:3',2'-d]imidazole relatedto protein pyrolysates have been studied for their effects onthe P-450 system of hepatic parenchyma and two dependent monoxidaseenzymes, zoxazolamine hydroxylase and dimethylnitrosamine-N-demethylase(DMN-d-ase). We found that the compounds can be divided intotwo groups. The first group consists of compounds which inhibitthe production of cytochrome P-450 and zoxazolamine hydroxylaseand induce the formation of DMN-d-ase; these compounds are knownto be powerful mutagens. The compounds of the second group,non-mutagens or weak mutagens, have an opposite effect: theyinduce the biosynthesis of cytochrome P-450 and zoxazolaminehydroxylase and repress the production of DMN-d-ase. These findingsare of interest since it is known that certain of the studiedcompounds can be found in human food.     

Changes in the Quantity and Activity of Cytochrome P-450 Isozymes in Primary Cultured Rat Hepatocytes

Hepatocytes from male Spragne-Dawley rats pretreated with a cytochrome P-450 inducer, 3-methoxy-4-aminoazobenzene (3-MeO-AAB), 3-methylcholanthrene (MC) or phenobarbital (PB), were cultured in vitro , and changes in the quantity and activity of microsomal cytochrome P-450 isozymes in the cells were determined by means of immunochemical methods and a bacterial mutation test, respectively. The results of enzyme-linked immunosorbent assay using monoclonal antibodies against rat P-450 isozymes - test using Salmonella typhimurium TA 98 and carcinogenic aromatic amines. These results indicate that microsomal cytochrome P-450c in primary cultured rat hepatocytes is more stable in culture, in terms of both quantity and activity, than cytochrome P-450d and P-450b/e.     

Human liver microsomal cytochrome P-450 enzymes involved in the bioactivation of procarcinogens detected by umu gene response in Salmonella typhimurium TA 1535/pSK1002

A total of 57 procarcinogens was examined for induction of umu gene response in the chimeric plasmid pSK1002, carried in Salmonella typhimurium TA 1535, after incubation with a series of human liver microsomal preparations which had been selected on the basis of characteristic levels of individual cytochrome P-450 (P-450) enzymes. The 18 most active compounds were selected and further analyzed using the umu gene response and correlative studies with a larger number of microsomal preparations, enzyme reconstitution studies involving purified enzymes, immunochemical inhibition, and patterns of stimulation and inhibition of catalytic activity by 7,8-benzoflavone. The results collectively indicate that 16 of these 18 most potent genotoxins examined are activated primarily either by P-450NF (the nifedipine oxidase) or P-450PA (the phenacetin O-deethylase). P-450NF appears to be the major enzyme involved in the bioactivation of aflatoxin B1, aflatoxin G1, sterigmatocystin, trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene, 6-aminochrysene, and tris-(2,3-dibromopropyl)phosphate in human liver. P-450PA appears to be the major enzyme involved in the bioactivation of 2-amino-3-methylimidazo[4,5-f]quinoline, 2-amino-3,5-dimethylimidazo[4, 5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-aminoanthracene, 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole, 2-aminofluorene, 2-acetylaminofluorene, 4-aminobiphenyl, 3-amino-1-methyl-5H-pyrido[4,3-b] indole, and 2-aminodipyrido[1,2-a:3',2'-d]imidazole. More than one enzyme appears to contribute significantly to the bioactivation of the other two compounds examined, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b] indole and 6-nitrochrysene. The literature suggests that the two human liver P-450s involved in activation of these 16 procarcinogens are highly inducible by barbiturates, macrolide antibodies, and certain steroids (P-450NF) and by smoking and ingestion of charcoal-containing food (P-450PA); noninvasive assays are available to monitor the function of both P-450NF and P-450PA.     

2-Amino-3,4-dimethylimidazo[4,5-f]quinoline induces and inhibits cytochrome P450 from the IA subfamily in chick and rat hepatocytes.

Several heterocyclic amines, found in cooked food, are powerful mutagens in the Ames Salmonella mutagenicity test system. One of these, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) is one of the most mutagenic chemicals tested in this assay. In primary cultures of chick and rat hepatocytes, MeIQ, by itself, induced cytochrome P450 from the IA subfamily but was a weak inducer compared to 3-methylcholanthrene. However, in both chick and rat hepatocytes in culture, MeIQ decreased the amount of 3-methylcholanthrene-induced ethoxyresorufin deethylase activity, which is catalyzed by cytochrome P450 IA. The protein moiety of cytochrome P450 IA was decreased at MeIQ concentrations of 2.5 micrograms/ml or greater in chick hepatocytes and 25 micrograms/ml in rat hepatocytes. In hepatic microsomes from methylcholanthrene-treated chicks and rats, MeIQ was a competitive inhibitor of both ethoxyresorufin deethylase activity, a reaction catalyzed mainly by rodent cytochrome P450 IA1, and uroporphyrinogen oxidation, a reaction catalyzed by rodent P450 IA2. In cultured chick hepatocytes, MeIQ also decreased cytochrome P450-mediated oxidation of uroporphyrinogen by intact cells. The ability of MeIQ to inhibit as well as to induce cytochrome P450s of the IA subfamily may be important in assessing the mutagenic and carcinogenic effects of MeIQ in mammals.     

Species difference among experimental rodents in the activity and induction of cytochrome P-450 isozymes for mutagenic activation of carcinogenic aromatic amines

The expressions of hepatic microsomal cytochrome P-450 isozymes in male rats, mice, hamsters and guinea pigs were studied comparatively with or without an ip injection of a cytochrome P-450 inducer. The activity and quantity of microsomal cytochrome P-450 isozymes were determined respectively by a bacterial mutation assay with Salmonella typhimurium TA98 and immunochemical assays using monoclonal antibodies against rat cytochrome P-450 isozymes. 3-Methoxy-4-aminoazobenzene (3-MeO-AAB), 2-amino-3-methyl-9H-pyrido[2,3-b]indole acetate (MeA alpha C) and 3-methylcholanthrene were used as cytochrome P-450 inducers, and 7 carcinogenic aromatic amines including 3-MeO-AAB and MeA alpha C were used as substrates for the mutation assay. By means of these assays, we examined the species differences among rodents in the activity and induction rate of hepatic cytochrome P-450 isozymes responsible for the mutagenic activation of carcinogenic aromatic amines.     

On the identity of the xenobiotic-metabolizing form(s) of cytochrome P-450 in endocrine organs

The adrenal cortex, the testes and the ovary metabolize polycyclic aromatic hydrocarbons (PAHs), e.g., 7,12-dimethylbenz[a]anthracene (DMBA). These activities have previously been shown to involve the cytochrome P-450 monooxygenase system [18-20]. In attempt to identify the form(s) of cytochrome P-450 involved, microsomes from these endocrine organs were subjected to SDS-gel electrophoresis, followed by immunochemical analysis using the Western blot technique. Antisera raised against the purified rat hepatic cytochrome P-450 isozymes a, b + e, c, d and PB-PCNE were tested. It was concluded that the electrophoretic mobilities of the immunoreactive bands obtained were not identical to the mobilities of the purified isozymes cytochromes P-450 a, b, c, d, e or PB-PCNE. These results indicate that the PAH-metabolizing monooxygenase(s) in these endocrine organs may involve a novel form(s) of cytochrome P-450.